Platelet-derived growth factor (PDGF) was originally isolated from platelet lysates and identified as the major growth-promoting activity present in serum but not in plasma. Two homologous PDGF isoforms have been identified, PDGF A and B, which are encoded by separate genes (on chromosomes 7 and 22). The most abundant species from platelets is the AB heterodimer, although all three possible dimers (AA, AB and BB) occur naturally. Following translation, PDGF dimers are processed into .apprxeq.30 kDa secreted proteins. Two cell surface proteins that bind PDGF with high affinity have been identified, .alpha. and .beta. (Heldin et al., Proc. Natl. Acad. Sci., 78: 3664 (1981); Williams et al., Proc. Natl. Acad. Sci., 79: 5867 (1981)). Both species contain five immunoglobulin-like extracellular domains, a single transmembrane domain and an intracellular tyrosine kinase domain separated by a kinase insert domain. The functional high affinity receptor is a dimer and engagement of the extracellular domain of the receptor by PDGF results in cross-phosphorylation (one receptor tyrosine kinase phosphorylates the other in the dimer) of several tyrosine residues. Receptor phosphorylation leads to a cascade of events that results in the transduction of the mitogenic or chemotactic signal to the nucleus. For example, in the intracellular domain of the PDGF .beta. receptor, nine tyrosine residues have been identified that when phosphorylated interact with different src-homology 2 (SH2) domain-containing proteins including phospholipase C-.gamma., phosphatidylinositol 3'-kinase, GTPase-activating protein and several adaptor molecules like Shc, Grb2 and Nck (Heldin, Cell, 80: 213 (1995)). In the last several years, the specificities of the three PDGF isoforms for the three receptor dimers (.alpha..alpha., .alpha..beta., and .beta..beta.) has been elucidated. The .alpha.-receptor homodimer binds all three PDGF isoforms with high affinity, the .beta.-receptor homodimer binds only PDGF BB with high affinity and PDGF AB with approximately 10-fold lower affinity, and the .alpha..beta.-receptor heterodimer binds PDGF BB and PDGF AB with high affinity (Westermark & Heldin, Acta Oncologica, 32: 101 (1993)). The specificity pattern results from the ability of the A-chain to bind only to the .alpha.-receptor and of the B-chain to bind to both .alpha. and .beta.-receptor subunits with high affinity.
The earliest indication that PDGF expression is linked to malignant transformation came with the finding that the amino acid sequence of PDGF-B chain is virtually identical to that of p28.sup.sis, the transforming protein of the simian sarcoma virus (SSV) (Waterfield et al., Nature, 304: 35 (1983); Johnsson et al., EMBO J., 3: 921 (1984)). The transforming potential of the PDGF-B chain gene and, to a lesser extent, the PDGF-A gene was demonstrated soon thereafter (Clarke et al., Nature, 308: 464 (1984); Gazit et al., Cell, 39: 89 (1984); Beckmann et al., Science, 241: 1346; Bywater et al., Mol. Cell. Biol., 8: 2753 (1988)). Many tumor cell lines have since been shown to produce and secrete PDGF, some of which also express PDGF receptors (Raines et al., Peptide Growth Factors and Their Receptors, Springer-Verlag, Part I, p 173 (1990)). Paracrine and, in some cell lines, autocrine growth stimulation by PDGF is therefore possible. For example, analysis of biopsies from human gliomas has revealed the existence of two autocrine loops: PDGF-B/.beta.-receptor in tumor-associated endothelial cells and PDGF-A/.alpha.-receptor in tumor cells (Hermansson et al., Proc. Natl. Acad. Sci., 85: 7748 (1988); Hermansson et al., Cancer Res., 52: 3213 (1992)). The progression to high grade glioma was accompanied by the increase in expression of PDGF-B and the .beta.-receptor in tumor-associated endothelial cells and PDGF-A in glioma cells. Increased expression of PDGF and/or PDGF receptors has also been observed in other malignancies including fibrosarcoma (Smits et al., Am. J. Pathol., 140: 639 (1992)) and thyroid carcinoma (Heldin et al., Endocrinology, 129: 2187 (1991)).
Although the contribution of PDGF in the progression of several proliferative disease states has been recognized (Heldin, EMBO J., 11, 4251 (1992); Raines et al., supra), there is a relative paucity of effective inhibitors of PDGF. The identification of novel antagonists of PDGF is therefore highly desirable. Currently, antibodies are the most potent antagonists of PDGF that have been described. Neutralizing antibodies to PDGF have been shown to revert the SSV-transformed phenotype (Johnsson et al., Proc. Natl. Acad. Sci. U.S.A., 82: 1721 (1985)) and to inhibit the development of neointimal lesions following arterial injury (Ferns et al., Science, 253: 1129 (1991)). Other inhibitors that have been described to date include suramin (an aromatic hexaanion, which also inhibits many other growth factors and is relatively toxic), and neomycin, which at high concentrations (5 mM) inhibits binding of PDGF BB to the .alpha.- but not the .beta.-receptor (Williams et al., J. Biol. Chem., 259: 5287 (1984); Vassbotn et al., J. Biol. Chem., 267: 15635 (1992)).
A method for the in vitro evolution of nucleic acid molecules with highly specific binding to target molecules has been developed. This method, Systematic Evolution of Ligands by EXponential enrichment, termed SELEX, is described in U.S. patent application Ser. No. 07/536,428, entitled "Systematic Evolution of Ligands by Exponential Enrichment," now abandoned, U.S. patent application Ser. No. 07/714,131, filed Jun. 10, 1991, entitled "Nucleic Acid Ligands," now U.S. Pat. No. 5,475,096 U.S. patent application Ser. No. 07/931,473, filed Aug. 17, 1992, entitled "Nucleic Acid Ligands," now U.S. Pat. No. 5,270,163 (see also WO Publication No. 91/19813), each of which is herein specifically incorporated by reference. Each of these applications, collectively referred to herein as the SELEX Patent Applications, describes a fundamentally novel method for making a nucleic acid ligand to any desired target molecule.
The SELEX method involves selection from a mixture of candidate oligonucleotides and step-wise iterations of binding, partitioning and amplification, using the same general selection scheme, to achieve virtually any desired criterion of binding affinity and selectivity. Starting from a mixture of nucleic acids, preferably comprising a segment of randomized sequence, the SELEX method includes steps of contacting the mixture with the target under conditions favorable for binding, partitioning unbound nucleic acids from those nucleic acids which have bound specifically to target molecules, dissociating the nucleic acid-target complexes, amplifying the nucleic acids dissociated from the nucleic acid-target complexes to yield a ligand-enriched mixture of nucleic acids, then reiterating the steps of binding, partitioning, dissociating and amplifying through as many cycles as desired to yield highly specific, high affinity nucleic acid ligands to the target molecule.
The basic SELEX method has been modified to achieve a number of specific objectives. For example, U.S. patent application Ser. No. 07/960,093, filed Oct. 14, 1992, entitled "Method for Selecting Nucleic Acids on the Basis of Structure," describes the use of SELEX in conjunction with gel electrophoresis to select nucleic acid molecules with specific structural characteristics, such as bent DNA. U.S. patent application Ser. No. 08/123,935, filed Sep. 17, 1993, entitled "Photoselection of Nucleic Acid Ligands" describes a SELEX based method for selecting nucleic acid ligands containing photoreactive groups capable of binding and/or photocrosslinking to and/or photoinactivating a target molecule. U.S. patent application Ser. No. 08/134,028, filed Oct. 7, 1993, entitled "High-Affinity Nucleic Acid Ligands That Discriminate Between Theophylline and Caffeine," describes a method for identifying highly specific nucleic acid ligands able to discriminate between closely related molecules, termed Counter-SELEX. U.S. patent application Ser. No. 08/143,564, filed Oct. 25, 1993, entitled "Systematic Evolution of Ligands by EXponential Enrichment: Solution SELEX," now U.S. Pat. No. 5,567,588 describes a SELEX-based method which achieves highly efficient partitioning between oligonucleotides having high and low affinity for a target molecule. U.S. patent application Ser. No. 07/964,624, filed Oct. 21, 1992, entitled "Methods of Producing Nucleic Acid Ligands" now U.S. Pat. No. 5,496,938 describes methods for obtaining improved nucleic acid ligands after SELEX has been performed. U.S. patent application Ser. No. 08/400,440, filed Mar. 8, 1995, entitled "Systematic Evolution of Ligands by EXponential Enrichment: Chemi-SELEX," describes methods for covalently linking a ligand to its target.
The SELEX method encompasses the identification of high-affinity nucleic acid ligands containing modified nucleotides conferring improved characteristics on the ligand, such as improved in vivo stability or improved delivery characteristics. Examples of such modifications include chemical substitutions at the ribose and/or phosphate and/or base positions. SELEX-identified nucleic acid ligands containing modified nucleotides are described in U.S. patent application Ser. No. 08/117,991, filed Sep. 8, 1993, entitled "High Affinity Nucleic Acid Ligands Containing Modified Nucleotides," that describes oligonucleotides containing nucleotide derivatives chemically modified at the 5- and 2'-positions of pyrimidines. U.S. patent application Ser. No. 08/134,028, supra, describes highly specific nucleic acid ligands containing one or more nucleotides modified with 2'-amino (2'-NH.sub.2), 2'-fluoro (2'-F), and/or 2'-O-methyl (2'-OMe). U.S. patent application Ser. No. 08/264,029, filed Jun. 22, 1994, entitled "Novel Method of Preparation of 2' Modified Nucleosides by Intramolecular Nucleophilic Displacement," describes oligonucleotides containing various 2'-modified pyrimidines.
The SELEX method encompasses combining selected oligonucleotides with other selected oligonucleotides and non-oligonucleotide functional units as described in U.S. patent application Ser. No. 08/284,063, filed Aug. 2, 1994, entitled "Systematic Evolution of Ligands by Exponential Enrichment: Chimeric SELEX" and U.S. patent application Ser. No. 08/234,997, filed Apr. 28, 1994, entitled "Systematic Evolution of Ligands by Exponential Enrichment: Blended SELEX," respectively. These applications allow the combination of the broad array of shapes and other properties, and the efficient amplification and replication properties, of oligonucleotides with the desirable properties of other molecules. Each of the above described patent applications which describe modifications of the basic SELEX procedure are specifically incorporated by reference herein in their entirety.
In the present invention, the identification of high-affinity nucleic acid ligands to PDGF is described. Specifically, single stranded DNA ligands and 2'-fluoropyrimidine RNA ligands to PDGF are described.